The present invention relates to systems for separating a sample fluid in a liquid chromatography column and directing the effluent from the column to a point of use, or point of further separation or detection and identification, particularly to a combination of a chromatography column and an electrophoresis apparatus for directing and conditioning the effluent for further analysis and still more particularly to a combination of a horizontal or radial flow chromatographic column and an electrophoresis apparatus utilizing at least one capillary tube and a valving arrangement for controlling the column effluent and buffer material passing therethrough.
In a liquid chromatography system, a sample fluid, followed by an elution fluid are injected into a separation column. The separation column contains a packing or separating medium or matrix, as well known in the art, which interacts with the various components of the sample fluid to be separated. The separation columns generally known in the art are of a cylindrical construction, the fluid flowing axially through the separating medium bed retained in the column. As the sample and elution fluids pass through the separating medium bed, the components or constituents of the sample fluid travel through the medium at different rates as a result of their differential interactions with the separation medium. Consequently, these constituents emerge separated (i.e., with different elution times and rates) in the outlet stream of the column. However, to increase the column output and to handle larger volumes, higher pressures have been found to be necessary.
More recently, the problem associated with high pressure usage, has been substantially resolved by utilizing liquid chromatographic columns witn a horizontal or radial flow through the separation medium in the column, which results in an increase in the output, using the same separation medium. Thus, higher flow rates at lower operating pressures can be achieved using the horizontal flow technique. These horizontal or radial flow liquid chromatography columns are described and claimed in copending U.S. patent application Ser. Nos. 794,727 filed Nov. 4, 1985 and 869,295 filed June 2, 1986, each in the name of Vinit Saxena.
In many applications, it is desirable to analyze and identify the chemical composition of one or more components of a sample fluid separated in a liquid chromatographic column. Such an analysis may be carried out, for example, in a mass spectrometer or by any other suitable detection device. When a mass spectrometer is used as the detection device, the fluid discharged from the liquid chromatography column must, in addition to being transported to the mass spectrometer, be rendered susceptible or amenable to the ionization thereof in the ion chamber of the mass spectrometer. Mass spectrometers are well known for their capabilities in analyzing and accurately identifying various chemicals but they also operate under high vacuum conditions, where the pressures are very low, of the order of 10.sup.-5 Torr or lower. The separated fluids discharged from the liquid chromatography columns are, most often, at atmospheric pressure or higher. In certain other applications, it would also be desirable for further separation and resolution of the effluent components, before the final analysis and identification.
Various methods and means have been designed and/or proposed for transporting the column effluent directly into the ion source of a mass spectrometer. Methods have also been proposed or designed for conditioning the column effluent for further separation and resolution of the effluent into various components, before being analyzed in the mass spectrometer while, at the same time, maintaining the integrity of the vacuum in the mass spectrometer. One such prior known means for the further separation and resolution of the components of the effluent and for conditioning it for entry into the mass spectrometer, if desired, is an electrophoresis apparatus.
Electrophoretic separations and techniques are known in the art and can provide very clean separations of many types of complex molecules in biological matrices. Typically, an electrophoretic chromatogram or electropherogram (these terms are used interchangeably herein) identifies multiple compounds as fairly discrete spots, bands or locations in an electrophoretic medium or identifies multiple compounds in an electrophoretic band or spot, but does not provide for structure elucidation or for the specific identification of individual compounds. Thus, the electrophoretic bands or spots must be prepared or conditioned for identification using a variety of transfer of staining techniques that are usually cumbersome and costly and identification is made only in terms of the electrophoretic mobilities of the unknowns in comparison with the mobilities of known standards, as is well known in the art.
More recent electrophoretic techniques utilize open tubular columns or capillaries, which allow individual derivatized or chemically tagged or isotopically labeled components to migrate past an on-line detector sensitive to or specific for the label, tag or isotope. Although this approach is sensitive, it is not specific and the compounds of interest may not be amenable to further derivatization and identification. Furthermore, dissimilar compounds may have identical or near identical migration rates and produced similar electropherograms and therefore, do not lend themselves to be differentiated.
Recently, it was discovered that when a high voltage is applied to a long, narrow bore capillary tube made of glass or quartz, an electrophoretic separation of organic, inorganic and biological compounds can be achieved. while this new capillary electrophoresis technique is being utilized for the separation and characterization of complex mixtures, it has poor sensitivity for structure elucidation and only chromatographic peaks are generated.
These prior problems of the capillary electrophoresis technique have been overcome by a new approach which utilizes capillary tubes but does not require fluorescent tags, and which operates in real time, and yields molecular weights, formulas and structural information about the compounds subjected to this new approach. This new approach is described and claimed in copending U.S. application Ser. No. 06/906,847, filed 09/15/86, 1986 in the name of Brian D. Andresen and Eric R. Fought and entitled "Electrophoresis-Mass Spectrometry Probe". With the new capillary electrophoresis technique, mixtures of chemicals, electrophoretically separated, can be directly identified by mass spectrometry.
As greater efforts are now being directed to the development of new analytical techniques and approaches, applicable to the emerging field of biotechnology, greater needs have arisen for faster and more accurate (more sensitive and specific) methods of separating biological fluids and for faster and quicker analysis of the components thereof, while minimizing the costs involved at the same time.
Therefore, it is an object of this invention to provide a means for economically separating fluids, especially biological fluids, into their various components and rapidly transporting and conditioning the components for analysis, purification or other points of use.
A further object of this invention is to provide a combined horizontal flow liquid chromatographic column and an electrophoresis apparatus for further separating and resolving the fluid components and for transporting and reducing the volume of fluids separated in the column for substantially simultaneous (real time) analysis thereof in a mass spectrometer.
Another object of the invention is to provide a means to combine a high performance liquid chromatography column, which uses readily replaceable or separation media, with a capillary tube type electrophoresis apparatus.
Yet another object of the invention is to provide a combination of a horizontal or radial flow chromatography column and a capillary tube-type electrophoresis apparatus connected via a valved coupling wherein the separated fluid components from the column are selectively directed into the electrophoresis apparatus for conditioning therein while being transported to a point of analysis or use.
Additional objects, advantages and novel features of the invention will be set fortn in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.